Xylanases are a group of enzymes with side commercial utility. A major application of xylanases is for pulp biobleaching in the production of paper. In addition, xylanases have been used as clarifying agents in juices and wines, as enzymatic agents in the washing of precision devices and semiconductors (e.g. U.S. Pat. No. 5,078,802), and they are also used for improving digestibility of poultry and swine feed.
In the manufacturing of pulp for the production of paper, fibrous material is subjected to high temperatures and pressures in the presence of chemicals. This treatment converts the fibers to pulp and is known as pulping. Following pulping, the pulp is bleached. Xylanase enzymes are used to enhance the bleaching of the pulp. The xylanase treatment allows subsequent bleaching chemicals such as chlorine, chlorine dioxide, hydrogen peroxide, or combinations of these chemicals to bleach pulp more efficiently. Pretreatment of pulp with xylanase increases the whiteness and quality of the final paper product and reduces the amount of chlorine-based chemicals which must be used to bleach the pulp. This in turn decreases the chlorinated effluent produced by such processes.
The most important chemical pulping process is kraft pulp. For kraft pulp, following pulping, and prior to the treatment of pulp with xylanase, the pulp is at about a temperature of 55-70° C. and at a highly alkaline pH (e.g. Nissen et al., 1992). A drawback of many commercially available wild-type xylanases, is that these enzymes exhibit an acidic pH optimum and a temperature optimum of about 55° C. Therefore, in order to effectively utilize xylanases for bleaching applications, the pulp must be acidified to a pH approximating the optimal pH for the specific xylanase used. In addition, the hot pulp must be cooled to a temperature close to the optimal temperature for enzymatic activity of the selected xylanase. Decreasing pulp temperatures for xylanase treatment decreases the efficiency of the subsequent chemical bleaching. Acidification of pulp requires the use of large quantities of acids. Further, the addition of acids leads to corrosion, which lessens the lifetime of process equipment. Thus, xylanases optimally active at temperatures and pH conditions approximating the conditions of the pulp would be useful and beneficial in pulp manufacturing.
Xylanases which exhibit greater activity at higher temperatures could be used to treat pulp immediately following the pulping process, without the need to cool the pulp. Similarly, xylanases which exhibit greater activity at higher pH conditions would require less or no acid to neutralize the pulp. The isolation of, or the genetic manipulation of, xylanases with such properties would provide several advantages and substantial economic benefits within a variety of industrial processes.
Several approaches directed towards improving xylanase for use in pulp-bleaching within the prior art include the isolation of thermostable xylanases from extreme thermophiles that grow at 80-100° C., such as Caldocelium saccharolyticum, Thermatoga maritima and Thermatoga sp. Strain FJSS-B.1 (Lüthi et al. 1990; Winterhalter et al. 1995; Simpson et al. 1991). However, these thermostable xylanase enzymes are large, with molecular masses ranging from 35-120 kDa (320-1100 residues), and exhibit a reduced ability to penetrate the pulp mass compared with other smaller xylanases which exhibit better accessibility to pulp fibers. In addition, some of the extremely thermophilic xylanases, such as Caldocellum saccharolyticum xylanase A, exhibit both xylanase and cellulase activities (Lüthi et al. 1990). This additional cellulolytic activity is undesirable for pulp bleaching, due to its detrimental effect of cellulose, the bulk material in paper. Furthermore, hyper-thermostable xylanase enzymes which function normally at extremely high temperatures have low specific activities at temperatures in the range for optimal pulp bleaching (Simpson et al. 1991).
A number of xylanases have been modified by protein engineering to improve their properties for industrial applications. For instance, U.S. Pat. No. 5,759,840 (Sung et al.), and U.S. Pat. No. 5,866,408 (Sung et al.) disclose mutations in the N-terminal region (residues 1-29) of Trichoderma reesei xylanase II (TrX). Three mutations, at residues 10, 27 and 29 of TrX, were found to increase the enzymatic activity of the xylanase enzyme at elevated temperatures and alkaline pH conditions.
U.S. Pat. No. 5,405,769 (Campbell et al.) discloses modification of Bacillus circulans xylanase (BcX) using site-directed mutagenesis to improve the thermostability of the enzyme. The site specific mutations include replacing two amino acids with Cys residues to create intramolecular disulfide bonds. In addition, specific residues in the N-terminus of the enzyme were mutated which were also found to further improve the thermostability of the enzyme. In in vitro assays, the disulfide mutants showed thermostability at 62° C., an improvement of 7° C. over the native BcX xylanase enzyme. However, these thermostable disulfide mutants showed no gain in thermophilicity in laboratory assays in subsequent studies (Wakarchuck et al., 1994). Mutations T3G (i.e. threonine at position 3 replaced with Gly; BcX xylanase amino acid numbering), D4Y(F) and N8Y(F) near the N-terminus of the BcX xylanase enzyme provided thermostability to 57° C., an increase of 2° C. over the native BcX (U.S. Pat. No. 5,405,769). However, the use of these enzymes within industrial applications still requires cooling and acidification of pulp following pretreatment, prior to enzyme addition. Therefore, further increases in thermostability, thermophilicity and pH optima are still required.
Turunen et al. (2001) discloses mutations (N11D, N38E, Q162H) of TrX II at positions 11, 38 and 162, complement similar disulfide bond (S110C/N154C) to improve the thermostability of the xylanase. However, these mutations including N11D also have an adverse effect on both the thermophilicity and the alkalophilicity of the xylanase, resulting in a decrease of enzymatic activity at higher temperatures and the neutral-alkaline pH, as compared to native TrX II.
There is a need in the prior art to obtain novel xylanases which exhibit increased enzymatic activity at elevated temperatures and pH conditions, suitable for industrial use. It is an object of the invention to overcome drawbacks in the prior art.
The above object is met by the combination of features of the main claim, the sub-claims disclose further advantageous embodiments of the invention.